Hygrolansamycins A-D, O-Heterocyclic Macrolides from Streptomyces sp. KCB17JA11

Six ansamycin derivatives were isolated from the culture broth of Streptomyces sp. KCB17JA11, including four new hygrolansamycins A-D (1-4) and known congeners divergolide O (5) and hygrocin C (6). Compounds 1-5 featured an unusual six-membered O-heterocyclic moiety. The isolation workflow was guided by a Molecular Networking-based dereplication strategy. The structures of 1-4 were elucidated using NMR and HRESIMS experiments, and the absolute configuration was established by the Mosher’s method. Compound 2 exhibited mild cytotoxicity against five cancer cell lines with IC50 values ranging from 24.60 ± 3.37 μM to 49.93 ± 4.52 μM.

Tandem mass-based dereplication is a common technique for screening known bioactive compounds, such as ansamycins, in complex microbial samples [10][11][12][13][14][15][16]. Global Natural Products Social Molecular Networking (GNPS), an open-access data-driven tandem mass spectral platform, is particularly well-suited for this purpose and is widely used. Previously, during our tandem-mass-based dereplication study of Streptomyces species, we discovered geldanamycin [10] and streptimidone [11] derivatives. In this study, we investigated the fermentation of Streptomyces sp. KCB17JA11, a geldanamycin producer, and using the GNPS system, we discovered new nodes, corresponding to divergolide O (5) [7] and hygrocin C (6) [5] with molecular weights of 513 and 509, respectively. Four other compounds were found to have the same molecular formula (C 31 H 39 NO 8 ) as divergolide O (5). Herein, we present four new stereochemical derivatives of 5, namely hygrolansamycins A-D (1-4).

Modified Mosher's Method
Compounds 1 and 4 (0.5 mg) was dissolved in anhydrous pyridine (1 ml), and a catalytic amount of dimethylaminopyridine (DMAP) was added. After 5 min of stirring, 25 μl of (R)-MPA-Cl was added, and the mixture was stirred at room temperature for 16 h. Repeat treatment of same method with (S)-MPA-Cl instead of (R)-MPA-Cl. Each mixture was subjected to semi-preparative reversed phase HPLC (column as above; flow rate 3 ml/min; 50-100% CH 3

Results and Discussion
Streptomyces sp. KCB17JA11 was cultured in YMG media at 28°C for 7 days, before the broth and mycelia extracts were partitioned using EtOAc. The EtOAc extracts were examined using ESI-ion trap-MS/MS and the data were processed into molecular networks using the GNPS platform (http://gnps.ucsd.edu) [13]. Among the nodes, the node shown in Fig. 1 was annotated through library searching, corresponding to divergolide O (5) and hygrocin C (6). The precursor ion at this node had an m/z value of 514. 3 [M+H] + . The High-performance liquid chromatography (HPLC) data and total ion current of the crude extract revealed the presence of six peaks eluting at 11.52, 11.71, 12.23, 12.65, 12.78, and 13.58 min, all of which had the same m/z values (514. 3 [M+H] + ) as the standard 5, which eluted at 12.65 min, as confirmed by retention time and MS/MS spectra comparison (Fig. 1). To obtain compounds 1-5, purification was performed using silica open-column liquid chromatography, reversephase MPLC, and semi-preparative HPLC. Thus, five compounds had m/z values of 514.3. Furthermore, 6 was isolated and identified from the same crude extract fractions (Fig. 2).
Hygolansamycin A (1) H y g o l a n s a m y c i n B ( 2) H y g o l a n s a m y c i n C ( 3) Hygrolansamycin A (1) was obtained as a yellow powder. Based on high resolution electrospray ionisation mass spectroscopy (HRESIMS) data, the molecular formula of 1 was assigned as C 28 H 35 NO 8 , having 12 degrees of   The ¹H-¹H correlation spectroscopy (COSY) spectrum, combined with Heteronuclear multiple bond correlation (HMBC) correlation analysis, established the presence of two spin systems; thus, fragments C-6/C-7/C-8(C-15/ C-16)/C-9/C-10/C-11/C-12(C-13) and C-2′′/C-3′′ were assigned. Key correlations from H-11 to C-5′′, H-3′′ to C-5′′ and C-6′′, and H-6′′ to C-3′′, C-4′′, and C-5′′ indicated the presence of two fragments linked via the ester moiety and the methylated position at C-6′′. Furthermore, six typical aromatic carbon signals at δ C 106.6 (C-1′), 151.2 (C-2′), 107.7 (C-3′), 126.4 (C-4′), 132.6 (C-5′) and 122.5 (C-6′), as well as HMBC correlations of NH/C-4′ and 1′′, H-3′/C-1′ and C-2′, H-1′/C-5′ and C-6′, suggest the presence of the 1-amino-3-hydroxybenzene moiety. Further analysis of HMBC correlations from H-1 to C-2, C-14, C-3, C-5, and C-5′, from H-4 to C-2, C-3, C-5, and C-6 and from H-6 to C-3, C-4 and C-5 suggested that compound 1 is an unusual six-membered O-heterocyclic ring (Fig. 3). Comparison of the planar structural data with those reported for divergolide O (5) revealed that 1 was closely related to 5, except for only relative configurations from H-2 to H-4 rotating-frame nuclear Overhauser effect correlation spectroscopy (ROESY) correlation (Fig. 2). In the previous report, the stereochemical study of O-heterocyclic ring moiety had been performed by NMR spectral analysis and X-ray crystallography in divergolide A [4]. In order to establish the relative configuration of O-heterocyclic ring moiety of compound 1, detailed analyses of 1 H NMR and ROESY data were performed. A large coupling constant ( 3 J = 14.5 Hz) between H-1 and H-2 showed the anti orientation for the two protons, whereas H-2 and H-14 was showed simply three bond coupling constant ( 3 J = 7.2 Hz). The ROESY correlation between H-2 (δ H 2.45) and H-4 eq (δ H 2.12) of 1 indicate that equatorial preferred for both H-2 and H-4 eq (Fig. 4A). On the other hand, the relative stereochemistry of the O-heterocyclic ring moiety in reported divergolide O (5) was descripted through NOE correlation between H-2 and H-4 ax [7]. In addition, the configuration of the C-3′′/4′′ double bond was assigned as E configuration because of the chemical shift of the allylic methyl group C-6′′ (δ C 13.1) [4]. The absolute configuration of C-11 was determine using a modified Mosher's method. Methoxyphenylacetic (MPA) esters were prepared by treating 1 with (R)-and (S)-MPA-Cl in anhydrous pyridine, yielding the corresponding (S)-and (R)-MPA esters 1a and 1b. C-11S configuration was established for 1 based on the Δδ [δ(S)δ(R)] values of the MPA esters (Fig. 4D). Furthermore, the coupling constant of J H11/H12 = 6.8 Hz in 1 indicate that the dihedral angle of these two protons was approximately 60° (Fig. 4C). In addition, the strong ROESY correlation between H-11 and H-12, but not between H-11 and the H-13 methyl protons of 1, indicated that H-11 was oriented anti to this methyl group (Fig. 4C). Thus, the relative configuration of C-1/C-2/C-5/C-11/C-12 was designated as 1S*/2S*/ 5S*/11S*/12S*. However, the relative configuration C-8 bearing the ethylated branch remained unknown because of the lack of relevant ROESY correlations, which was attributed to the flexibility of that portion of the ansa macrolactam. To confirm the configuration of C-8 stereocenter in 1, using chiroptical analysis involving circular dichroism (CD) spectroscopy. The previously reported CD spectra of 5 and divergolides were elucidated as 1S/2R/ 5S/8R/11S/12S by contrasting with the mirror image and confirming a closely similar Cotton effect to divergolide A [4,6,7]. Further, the experimental CD spectra of 1 show strikingly similar Cotton effect from 5 over the entire wavelength range (Fig. 5). Therefore, the absolute configuration of 1 was established 1S/2S/5S/8R/11S/12S. Thus, compound 1 was named as hygorlansamycin A.
Hygrolansamycin B (2) was isolated as a yellow powder. According to HRESIMS data the molecular formula of 2 was determined to be C 28 H 35 NO 8 , which is the same as that of 1. The orientation of the C-3′′/4′′ double bond was the only difference between the structures of 1 and 2. The relative downfield shift of the C-6′′ allylic methyl group (δ C 13.1, δ H 1.86 in 1 and δ C 20.4, δ H 1.99 in 2) was used to deduce that the C-3′′/4′′ double bond was in Z configuration. Thus, 1 is a geometric isomer of 2 [4]. Hygrolansamycin C (3) was isolated as a yellow powder. The molecular formula was determined to be C 28 H 35 NO 8 on the HRESIMS data, which is the same as that of 2. The high similarity between the 1 and 2D NMR spectra of 2 and 3 indicated that they shared the same planar structure. The ROESY spectrum correlation between H-2 and H-4 ax (δ H 2.98) indicates that O-heterocyclic ring is axial preferred for both H-2 and H-4 ax (Fig. 4B) [6,7]. Furthermore, the 1D NMR chemical shifts of C-14 (δ C 15.3, δ H 1.29 in 2 and δ C 9.9, δ H 0.89 in 3) were shown relative configuration of C-2, which is opposite in between 2 and 3 [17]. Thus, 3 is a stereoisomer of 1.
Hygrolansamycin D (4) was isolated as a yellow powder. The HRESIMS data indicated that the molecular formula of 4 was C 28 H 35 NO 8 . The high similarity between the 1 and 2D NMR spectra of 1 and 4 indicated that they shared the same planar structure. The ROESY spectrum ( Fig 4B) and chemical shifts of C-14 (δ C 15.5, δ H 1.28 in 1 The O-heterocyclic ring moiety of 5 [7] was present in the four hygrolansamycin congeners 1-4. However, closer examination of the NMR data revealed some variations at position C-2 in the O-heterocyclic ring. The H-1 signals appeared as doublets in the 1 H NMR spectra of 3 and 4, but not in those of 1 and 2 [7,17]. Therefore, H-1 and H-2 are anti orientated in 1 and 2, while in 3 and 4, they are syn. Furthermore, the C-3′′/C-4′′ double bond shifted to the β, γ-position, rather than the typical α, β-position, which results from traditional syn elimination of water from a β-hydroxy function. From the relatively shielded chemical shifts of C-6′′ (δ C 13.1 in 1 and δ C 13.2 in 4) of compounds 1, 4, and 5, C-3′′/4′′ was E configuration, whereas the C-6′′ (δ C 20.4) of 2 and 3, C-3′′/4′′ was determined to be Z configuration [6]. Except for the C-3"/4" double bond, other chromophores have little effect on the Cotton effects. Compounds 1, 4, and 5 with E forms show different Cotton effects from compounds 2 and 3 in the 200-280 nm wavelength range, but all spectra are very similar at wavelengths above 290 nm. In addition, reported divergolides E, G, H, and O (5) show a positive Cotton effects at 260 nm, while 2 and 3 with the Z form show a lower negative Cotton effects at 230-260 nm, similar to those of reported divergolides A and F (Fig. 5) [4,6,9]. Although the O-heterocyclic moiety, C-3′′/4′′ double bond and isobutyl moiety of divergolides may serve as additional chromophores, the hygrolansamycins found in this study showed similar CD spectral patterns. These results show that the differences in chromophore of O-heterocyclic ring moiety does not affect the CD spectrum patterns of compounds.